The goal is to evaluate the use of DNA probes for transient RNA molecules in testing the susceptibility of Mycobacterium tuberculosis to antibiotics. We will evaluate two types of transient RNA as indicators of antibiotic-resistant metabolic activity in cultures or clinical samples challenged briefly with antibiotics. First, we will clone, sequence, and develop DNA probes for M. tuberculosis ribosomal RNA (rRNA) precursor sequences. Second, we will use differential or subtractive screening techniques to identify specific messenger RNA (mRNA) sequences which, due to regulatory response to antibiotic challenge and mRNA turnover, are depleted in susceptible cells exposed to isoniazid, ethambutol, or streptomycin. DNA probes for these mRNAs will be developed and tested. Probes for both types of transient RNA will be combined with probes for species-specific nucleic acid sequences to result in assays that can rapidly and simultaneously detect M. tuberculosis and test its susceptibility to antibiotics. As a functional assay for antibiotic effectiveness, this general approach will be able to detect antibiotic resistance rapidly and specifically, regardless of the genetic basis for resistance. This work will also provide us with useful data on the biology of growth and ribosome assembly in M. tuberculosis, and may add to our understanding of the modes of action of important antituberculosis drugs.